Superoxide dismutase (SOD) occurs naturally in the biosphere or living world extensively and is known as an enzyme capable of eliminating superoxide anion radicals (hereinafter referred to briefly as "radicals") which are metabolites of oxygen. SOD is found in all and every living beings or organisms that require oxygen for growth, such as animals, plants and microorganisms.
It has been discovered and confirmed in man that SOD exists in three different types; SOD containing copper and zinc, SOD having manganese and extracellular SOD being present outside cells, among which the type containing copper and zinc is widely known to the general public. Reports have been made that such different types of SOD are effective for rheumatism and arthritis deformans, and in expectation of preventing injuries by ischemia- recirculation, for example, clinical trials are currently under way but in most cases through administration of SOD in the unmodified state. In the light of the fact that such types of SOD show a half-life in the blood as short as some minutes after administration, it cannot be said that SOD is allowed to produce its own effects to the maximal extent; in other words, the efficacy testing has been conducted only on a limited scope.
This invention is concerned with a process which can permit modified superoxide dismutases with a high degree of purity having a markedly elongated blood half-life and consequently finding a wide range of clinical application as a drug substance to be produced on a commercial scale in simplified manners and in increased yields. Heretofore, a great variety of chemical modifications have been performed in order to provide SOD with a prolonged half-life in the blood. For example, modification of SOD was made with high-molecular-weight dextrin (W. F. Petrone et al., Proc. Natl. Acad. Sci. U.S.A., 77, 1159 (1980)), polyethylene glycols (The Japanese Unexamined Patent Publication Nos. 249,388/61, 115,280/62 and 245,671/63, and a report by Charles O. Beauchamp et al. (Analytical Biochemistry, 131, 25-33 (1983)) or inulin (The Japanese Unexamined Patent Publication No. 32,826/58). Nevertheless, the said modified SODs as well as the above-mentioned processes for the production of such SODs suffer from various disadvantages to be described below, resulting in failure to solve the problems satisfactorily.
The above-described SOD derivatives have all been developed for the purpose of preventing oxidative tissue injuries in the living body through intravenous and intramuscular administration. Except as stated in the above report by Charles O. Beauchamp et al., all of such derivatives are the high-molecular-weight modified SODs that are produced by use of activated modifying agents having two functional groups with the result that two molecules of SOD are introduced, and consequently, they are provided with an extremley extended half-life in the blood; they present the disadvantage that they remain in the living body for a MUCH TOO long period of time, although they offer the advantage of having a longer blood half-life than unmodified SOD. Beauchamp et al., in their report mentioned previously, described that they succeeded in making improvements on such disadvantage through the activation of N,N'-carbonyldiimidazole (CDI) to thereby supply polyethylene glycol (PEG) with only one functional group. However, the method suffers from the defects that the treatment batch size is too small to conduct the commercial production of modified SOD, particularly the large-volume production of the same for pharmaceutical uses, and that the treatment requires a prolonged length of time.
Taking into consideration the above situation, this invention has been devised after intensive investigation into the large-volume production of SOD modified with polyethylene glycol (hereinafter referred to briefly as "PEG-SOD") or SOD modified with polyoxyethylene glycol.polyoxypropylene glycol.polyoxyethylene glycol (hereinafter referred to briefly as "PEG-PPG-SOD") to thus utilize them in the application fields as pharmaceuticals.
The present inventors, in the course of research activities carried out on the application of proteins to pharmaceuticals, found that prolongation of blood half-lives of proteins can permit the application scope of proteins to be maximized, and have already filed an application for patent covering the efficacy-sustainable type of proteins as per Japanese Unexamined Patent publication No. 59029/59. Unmodified superoxide dismutase (SOD), that is usable in this invention, has in recent years been attracting enhanced attention as a pharmaceutical, and vigorous clinical studies are under progress with SOD.
Taking notice of the facts that unmodified SOD shows a blood half-life as short as some minutes in rats and that SOD elicits drug efficacy at by far larger doses than generally considered for the enzymatic proteins for medicinal uses, however, the present inventors have conducted repeatedly intensive research on a simplified method, as a means of eliminating such defects, of producing high-purity modified dismutase in improved yields that can realize the large-volume treatment. As described in the above, Beauchamp et al. reported the SOD modified with PEG activated by CDI but their produciton method is encountered with difficulties in conducting into practice for large-scale production in terms of facilities.